1. Field of the Invention
This invention provides a novel vector and method of using such vector, comprising recombinant entomopoxviruses, for delivery of genes of biological significance for expression in vertebrates, including humans.
2. Background
A number of heterologous gene delivery systems have been evaluated in vertebrate hosts. These include vaccinia virus recombinants, recombinant retroviral vectors, naked DNA vectors, biolistic gene delivery, or delivery of recombinant DNA constructs via liposomes or other transfection enhancing means. Each of these known methods have advantages and disadvantages. Accordingly, there is an ongoing need in the art for new gene delivery vectors and methods, and this invention provides a further option for those skilled in the art, namely, the use of recombinant entomopoxvirus vectors for delivery of heterologous genes for expression in vertebrate cells.
To date, there does not appear to have been any disclosure or suggestion in the art that entomopoxviruses (EPVs) could be used to deliver and express genes of biological significance in vertebrate hosts. EPVs are poxviruses of insects and the most distant relatives of the more commonly studied vertebrate orthopoxviruses vaccinia, (VV) and cowpox virus (CPV). Orthopoxviruses are the only mammalian DNA viruses which replicate in the cytoplasm. To achieve this, the virus must synthesize all the enzymes necessary to transcribe and replicate the viral DNA within the cytoplasm.
Poxviruses comprise two sub-families, the Chordopoxvirinae (vertebrate viruses) and the Entomopoxvirinae (insect viruses; Arif, 1995; Moyer, 1994). Vaccinia is the prototypic vertebrate poxvirus. The Entomopoxvirinae (EPVs) were only recently discovered (1963) but are now known to be distributed world-wide and productively infect primarily Orthoptera, Diptera, Coleoptera and Lepidoptera orders of insects. The prototypical EPV is the lepidopteran virus isolated fromAmsacta moorei (AmEPV) which grows well in cultured insect cells.
The orthopoxviruses and the EPVs have some features in common, yet are quite distinct. Similarities include: morphology, a large linear double stranded genome (190 kb for VV, 225 kb for AmEPV), common sequence motifs (that in the vertebrate poxviruses are associated with transcriptional regulation), non-spliced transcripts, a cytoplasmic site of replication, and production of cytopathic effects in their natural host cells. Differences include: the G+C content of the viral DNA (37% for VV, but only 18% for AMEPV); optimal growth temperatures (26.degree. C. for AmEPV, 37.degree. C. for VV); and host range (AmEPV does not replicate in vertebrate cells and VV does not replicate in insect cells), although both viruses enter their non-permissive cells, (Langridge, 1983, and FIG. 1 herein). It has recently been observed that poxvirus promoters are not universally conserved as there are at least some late AmEPV promoters which are specific for the insect environment (Hall et al., 1996).
In U.S. Pat. No. 5,476,781, and foreign equivalents thereof (WO 92/14818 and WO 94/13812), entomopoxvirus spheroidin or thymidine kinase sequences and non-entomopoxvirus vectors containing these sequences were disclosed. However, these references neither disclose nor suggest a recombinant entomopoxvirus that is capable of acting as a vector for expression of an heterologous gene in a vertebrate cell. If anything, these publications tend to teach away from such a conclusion in that it is clearly stated that the entomopoxviruses are unable to replicate in vertebrate cells. Therefore, from these publications, one of ordinary skill in the art would be taught that a viral vector containing entomopoxvirus sequences would need to be a virus other than an entomopoxvirus to be useful for delivery of heterologous sequences to vertebrate or mammalian cells in vitro.
Entomopoxviruses are only known to productively infect and kill insects (Granados, 1981). No changes in cellular morphology by phase contrast microscopy were reported when Amsacta moorei entomopoxvirus (AMEPV, a virus isolated originally from the red hairy caterpillar) is used to infect mouse L-929 cells at a multiplicity of infection (m.o.i.) of 10 plaque forming units (p.f.u.) per cell.
Langridge (1983) demonstrated that AmEPV was able to enter a mammalian cell, but was reported to be unable to replicate or express its own proteins, as the gross .sup.35 S-methionine pulse-labeled protein pattern of AmEPV-inoculated L-929 cells was identical to the protein pattern from uninoculated cells.
Granados (1981) reported that there was no evidence of a productive AmEPV infection in a human (HeLa) cell line, and that the host range of AmEPV is restricted to insects. In a typical vertebrate poxvirus infection of a vertebrate cell, there is a rapid shut-down of host cell protein synthesis. However, as disclosed herein, vertebrate cells contacted with AmEPV failed to demonstrate this shut-down in protein expression.
In U.S. Pat. No. 5,338,679, Yuen and Arif disclosed vertebrate poxvirus expression vectors capable of expressing heterologous genes under the control of an entomopoxvirus gene promoter. The patentees mistakenly believed that the entomopoxvirus promoter they were using was the spheroidin promoter, but have since publicly acknowledged that the promoter they used was in fact the entomopoxvirus fusolin promoter. Thus, the Yuen and Arif patent discloses vertebrate poxviruses wherein expression of the heterologous gene is driven by what turns out to be the entomopoxvirus fusolin promoter. Accordingly, while this publication established the possibility of using an entomopoxvirus promoter to drive expression of a gene in a vertebrate, there was no disclosure or suggestion that entomopoxvirus itself could be used as the recombinant viral vector for delivery of and subsequent heterologous gene expression in a vertebrate system.
Paoletti, U.S. Pat. No. 5,174,993, disclosed a recombinant avipox virus in which an early entomopoxvirus promoter, referred to in that patent and herein as the AmEPV 42k promoter or as the EPV early strong promoter (esp), was used to drive expression of heterologous genes in a vertebrate viral vector. Thus, like the Yuen and Arif patent, this patent establishes the possibility of using an entomopoxvirus promoter to drive expression of a gene in a vertebrate. However, also like the Yuen and Arif patent, there is no disclosure or suggestion that entomopoxvirus itself could be used as the recombinant viral vector for delivery and subsequent heterologous gene expression in a vertebrate system.
Tine et al., (1996) disclosed an expression system in which the EPV 42k promoter was used to prepare a malaria vaccine in a recombinant vaccinia virus vector.
Dall et al., WO 93/25666, and EP 646172, published on Apr. 5, 1995, disclosed recombinant entomopoxviruses for use in the control of insect pests or for production of recombinant proteins in cell culture. This publication notes that an advantage of EPV's for recombinant production of proteins is that the "EPV's do not necessarily cause lysis of the infected cell, thereby offering potential for long term persistent infection of large scale cell culture." However, there is no disclosure or suggestion that the disclosed recombinant entomopoxviruses could be used in vitro in vertebrate cells or in vivo as a vector for delivery and subsequent heterologous gene expression in vertebrates.
Palmer et al. (1995), as with the Dall et al. PCT publication, disclosed genetic modification of various entomopoxvirus genomes. However, there is no disclosure or suggestion that the thus modified entomopoxviruses could serve as a recombinant viral vector for delivery and subsequent expression of an heterologous gene in a vertebrate.
Several publications have emerged showing that baculovirus, an insect virus unrelated to the entomopoxviruses, is able to deliver genes for expression in vertebrate cells (Hofman et al., 1995; Boyce and Bucher, 1996). However, the baculovirus system depends on the vertebrate cell's transcriptional machinery for heterologous gene expression. In addition, these references teach nothing with respect to EPV interactions with vertebrate cells and therefore stand apart from the instant disclosure of recombinant entomopoxviruses for delivery and subsequent expression of foreign genes in vertebrate cells.